废水生物处理过程中微生物胞外抗性基因的存在及水平转移机制

Environmental antibiotic resistance genes (ARGs) consist of both intracellular (iARGs) and extracellular ARGs (eARGs). The eARGs are likely more persistent and can spread via horizontal gene transfer under certain environmental conditions. However, up to date, very little is known about eARGs due to the difficulty in extraction and purification of extracellular DNA (eDNA). Considering that municipal wastewater treatment plants (MWTPs) are important sources of environmental ARGs with high content of eDNA in activated sludge, this work aims to investigate the occurrence and gene transfer of eARGs in the activated sludge process. First, a reliable method for extraction and purification of eDNA in activated sludge is developed for exploring the distribution and abundance of eARGs in different treatment units of MWTPs, aiming to reveal the fate of eARGs in MWTPs. In laboratory conditions, the effects of bioreactor operation parameters on the eARGs abundance in the presence and absence of antibiotic selection pressure are determined, and the key operation parameters are also identified. Thereafter, we study the persistence and gene transfer potential of exogenous eARGs as well as determine the microbial composition of activated sludge and the abundance of iARGs and integron. The internal relation between eARGs and microbial composition, iARGs, and integron is built based on above results. This research will provide helpful information for revealing the gene transfer mechanism of eARGs in wastewater biological treatment system.

环境中抗生素抗性基因（ARGs）的存在形态包括胞内ARGs和胞外ARGs，胞外ARGs在一定条件下可持久性存在并发生水平转移，但因为胞外DNA提取难度较大，目前有关胞外ARGs的研究仍然很少。针对城市污水厂是环境ARGs的重要污染源，且含有大量胞外DNA，本项目拟系统开展活性污泥胞外ARGs分布及水平转移研究。首先拟建立活性污泥胞外DNA的提纯方法，明确污水厂不同处理单元中胞外ARGs的存在水平，揭示其在污水厂中的归趋。在实验室条件下，考察抗生素存在与否时，反应器工艺参数对活性污泥胞外ARGs丰度的影响，识别关键影响工艺参数；在此基础上，探明外源胞外ARGs的持久性和水平转移潜力，同时分析活性污泥微生物群落结构和胞内ARGs、可遗传元件整合子的丰度，建立胞外ARGs与微生物群落结构、胞内ARGs、整合子的内在联系。该研究对于揭示胞外ARGs在废水生物处理过程中的传播扩散规律具有指导意义。

环境中抗生素抗性基因（ARGs）的存在形态包括胞内ARGs和胞外ARGs，胞外ARGs在一定条件下可持久性存在并发生水平转移，但因为胞外DNA提取难度较大，目前有关胞外ARGs的研究仍然很少。针对城市污水厂是环境ARGs的重要污染源，且含有大量胞外DNA，本项目系统开展了活性污泥胞外ARGs分布、水平转移以及控制方法研究。我们首先建立了不同赋存形态（胞内、胞外聚合物EPS、胞外游离）ARGs的提取方法，发现热提法（60°C、60 min）可有效提取活性污泥EPS中的ARGs，其存在丰度远远高于胞外游离ARGs；接着我们探究了EPS和胞外游离ARGs在污水厂不同处理单元中的削减规律，并利用16S rRNA测序和相关性分析揭示了EPS中ARGs潜在的宿主可能来源于Proteobacteria、Bacteroidetes；我们探究了胞外ARGs的环境持久性及其水平转移潜力，发现EPS中的ARGs可以持久性的存在并且具有很强的转化能力。为了降低胞外ARGs的耐药性传播风险，我们开发了混凝-微滤集成工艺，不仅可实现总ARGs和胞外ARGs的高效削减，还能有效减缓膜污染。研究成果为城市污水处理厂中微生物耐药性的传播和控制提供了理论依据。